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Reference materials for clinical analysis

Good health care requires reliable measurements of health status markers, mainly in blood samples. Such In-Vitro Diagnostics measurements need to be comparable over different hospitals and countries, and over time. The JRC performs pre-normative...

The measurement of biomarkers, mainly in blood, is performed in many thousands of hospital laboratories. The results are used to monitor health status, screen for disease, support diagnosis, and monitor the effect of therapies for diseases.

However, measuring biomarkers reliably is not straightforward. The results can be influenced by the calibration of the instrument, chemical instability, the selectivity of a particular method and eventually by the patient's genotype. A lack of harmonisation makes it impossible to compare test results obtained at different hospitals or even the same hospital over a period of time – a situation which is exacerbated by increased levels of population migration and mobility.

Making laboratory results comparable requires standardisation, using reference materials and methods. The need for standardisation and traceability is taken up in the EU Directive on in Vitro Diagnostic Medical Devices (IVD-MD). In Vitro Diagnostics manufacturers can make use of JRC’s standardisation tools in order comply with the requirements of the directive.

Measurement standards accepted worldwide

The JRC has the expertise to develop reference materials, but also coordinates the input from the clinical chemistry profession, the In Vitro Diagnostics industry and hospital clinicians. The close collaboration with specialists in the field ensures that the reference materials produced are the best suitable for clinical practice and patient outcome. Standardisation is both a scientific and a consensus process. Therefore the independent position of the JRC helps in developing reference systems that are generally accepted.

Support to the development of legislation and guidance documents

The need for standardisation and traceability is taken up in EU legislation. The the EU Directive on In Vitro Diagnostic Medical Devices (IVD-MD) (Directive 98/79/EC) is currently under revision, and the JRC contributed to the drafting of a proposal for a new regulation on in-vitro medical devices. The proposed regulation aims to ensure the smooth functioning of the internal market and a high level of protection of human health and safety.

It is also in this context that the JRC contributes to documentary standards and guidance developed by international initiatives like the International Standards Organisation (ISO), the International federation for Clinical Chemistry and Laboratory Medicine (IFCC) and the Joint Committee for the Traceability of Laboratory Medicine (JCTLM).

Building on a long tradition for the standardisation of the measurement of plasma proteins and enzymes

A major breakthrough in standardising plasma protein measurements was made in the 1990s when the European Commission's Bureau Communautaire de Références (BCR), in collaboration with the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC), released the stable and reliable reference material BCR-470, later re-named ERM-DA470/IFCC, certified for 15 proteins. Since then, the JRC has developed and released a range of clinical reference materials for proteins like C-reactive protein and cystatin C – markers for inflammation and for the functioning of the kidney, respectively. A reference material for 12 protein markers (called ERM-DA470k/IFCC) has been produced to ensure continuity in the measurement of the main plasma proteins like albumin and the antibodies IgG and IgM.

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In the same way, the standardisation of the measurements of the catalytic activity concentration of enzymes is a very demanding task in clinical chemistry. Enzymes are routinely measured as biomarkers for liver, kidney, cardiac and other functions. The catalytic activity of an enzyme is a property measured under specific experimental conditions. Therefore, the possibility of defining a single and universally recognised routine procedure was raised in the seventies. However, the use of a common procedure was not feasible due to the difficulty to reach a consensus between professionals on measurement and enzyme reaction conditions. The calibration of routine procedures using validated calibrants, traceable to a reference measurement procedure, seemed to be the best approach. Therefore, IFCC published a series of reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C. Since then, the JRC is developing certified reference materials (CRMs) used as quality control materials for the IFCC reference procedure. They cover, for example, the measurement of aspartate aminotransferase (measured for the evaluation of the liver function) and creatine kinase (marker for myocardial infarction and muscular dystrophy). These CRMs are, together with the reference measurement procedures, key components of the Reference Measurement Systems for reliable diagnostics of enzymes.

The JRC's reference materials for human plasma proteins and enzymes are the de facto standards that enable laboratories worldwide to use common reference ranges in diagnosis and to compare results between hospitals and over time. The process leading to the standardisation of plasma protein measurements is reviewed in Standardising plasma protein measurements worldwide: a challenging enterprise.

Supporting the early diagnosis and monitoring of major chronic diseases

Chronic diseases like autoimmune diseases, diabetes and Alzheimer disease are not only devastating for the patients and their families, but also have a major societal cost. The early diagnosis of such diseases is important for the development and implementation of treatments. However, many of these diseases are difficult to diagnose, particularly in early stages. Reliable biomarker measurements could make a big difference. JRC coordinates, together with the International Federation for Clinical Chemistry and Laboratory Medicine, the research required for the standardisation of the relevant biomarkers. Reference materials are being developed for biomarkers for diabetes (HbA1c) autoimmune diseases like vasculitis, rheumatoid arthritis and lupus, as well as for Alzheimer disease.

The JRC approach for the standardisation of complex biomarkers is described in the article Standardisation of protein biomarker measurements: Is it feasible?

Developing the bioanalysis methods that form the basis for stable reference systems

The reference methods and reference materials developed by the JRC form the basis for reliable and stable clinical measurements. Therefore these need to be developed at a level of quality well beyond what is required in routine analysis. This requires research into the processes underlying the measurements, and the rigorous validation of methods developed. This may seem a slow process, but this is one of the fields where time is required in order to achieve the necessary quality. An example is a method for the quantification of protein calibrants by amino acid analysis. JRC participates in ring trials with metrology institutes from all over the world in order to study how these methods can still be further improved.

Worldwide first certified reference materials for monitoring of leukaemia - ERM-AD623

Chronic myelogenous leukaemia (CML) is a cancer of the white blood cells. In the EU about 6200 new patients per year are diagnosed and CML mainly affects adults. CML can currently not be cured, but kept under control with specific enzyme inhibitors. Regular monitoring of the CML patient is needed to ensure optimal treatment of the patient.

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For the monitoring the number of specific genetic fragments, the BCR-ABL1 transcripts, is measured. Until now, the calibration of that monitoring method has been differently performed in various laboratories which caused non-comparable results. Therefore, a certified reference material (CRM) has been developed making use of specifically tailored plasmids suitable for the calibration of BCR-ABL1 measurements. The standardisation of the quantification of the copy numbers of BCR-ABL1 and the control gene transcripts will improve the treatment of the patient. In essence, this new CRM is an essential tool to correctly assess the human response to leukaemia treatment and to detect a relapse of individual patient early.

The CRM is a plasmid containing selected desoxyribonucleic acid (DNA) fragments specific for the transcript of breakpoint cluster region gene (BCR), the transcript of the glucuronidase beta gene (GUSB) and the fusion transcript from the BCR gene and the c-abl oncogene 1 (BCR-ABL b3a2). The CRM, coded as ERM®-AD623a–f, has been certified for the copy number ratio of these specific fragments per plasmid and for the copy number concentration of the plasmid per volume of solution. ERM-AD623 was prepared according to ISO Guide 34 and characterised by expert laboratories of demonstrated competence using the most advanced counting method for small DNA amounts, the so-called digital polymerase chain reaction. Uncertainties of the certified values were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement (GUM)

The preparation and certification of ERM®-AD623 is described in detail in the corresponding certification report.

RM for clinical analysis

RM for clinical analysis